α-Glucosidase inhibitive diarylheptanoids from Ottelia acuminata var. acuminata, a traditional vegetable of Bai Nationality in Yunnan

Diabetes is an urgent health issue characterized by ethnic and regional variations, and is inseparable from the different dietary habits. It is worthy to note that the incidence of diabetes in Bai nationality has been reported to be much lower than Han in China. As a daily vegetable of Bai, the phytochemical and antidiabetic study of Ottelia acuminata var. acuminata had not been carried out. In this study, 41 metabolites with diverse diarylheptanoid (six new ones, Otteacumienes A–F), flavone, sesquiterpenoid, coumarin, lignan, polyacetylene, and alkaloid skeletons were characterized from O. acuminata var. acuminata. Among them, the racemic nature of 3 was characterized by chiral resolution and calculated ECD methods. The biological study revealed diarylheptanoids showed significant α-glucosidase inhibitory activities with 5 as the most effective one (60-fold stronger than acarbose). Molecular docking studies indicated that these structures have different binding cavities with acarbose. This study demonstrated that O. acuminata var. acuminata might correlated with the low incidence diabetes of Bai and the diarylheptanoids may have potential therapeutic value for diabetes mellitus. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-022-00341-4.


Introduction
The latest research from IDF (International Diabetes Federation) revealed that about 8.8% of the world's population suffers from diabetes disease. Among them, type 2 diabetes (T2DM) is the most common type, accounting for more than 90% of all diabetic cases worldwide [1]. Treatment of T2DM requires both diet and exercise to address the overweight or obesity. A range of combination therapy options were available for T2DM such as sulfonylureas, α-glucosidase inhibitors (AGI), thiazolidinediones, dipeptidyl peptidase 4 (DPP-4) inhibitors, glucagon-like peptide 1 (GLP-1) agonists, and sodiumglucose co-transporter 2 (SGLT-2) inhibitors [2]. Among them, α-glucosidase is one of the most important digestive enzymes in the human body involved in the final step of carbohydrate digestion, and its inhibitors contributes to control postprandial glucose for diabetes treatment [3]. However, the common side effects of flatulence, diarrhea, and hepatotoxicity for the clinical α-glucosidase inhibitors (including acarbose and miglitol) encouraged us to find new type of inhibitors with high safety and efficiency [4].
It's well-know that the development of diabetes is closely relevant to the dietary and lifestyle. Interestingly, the investigation of the incidence of diabetes in China has showed the characteristics of ethnic and regional variations [5], especially for the Bai nationality (7.83%) in contrast to the Han nationality (11.83%) in rural Yunnan [6]. As one of the major ethnic minorities in Yunnan, the Bai nationality has a long history and splendid culture, most of whom live in Dali Bai Autonomous Prefecture [7]. Notably, O. acuminata var. acuminata had long been used as a traditional daily vegetable in Bai as record in the "Textual Research on Reality and Titles of Plants" published in 175 years ago [8].
As an edible unique plant in China, Ottelia acuminata var. acuminata belongs to the genus Ottelia of the Hydrocharitaceae family, and mainly distributed in Yunnan Province [9]. This plant is also a famous aquatic ornamental plant, attracting millions of tourists to Erhai and Lugu Lakes every year during the blooming period from May to October. Additionally, the high requirements for water quality could be applied to monitor water pollution and environmental protection [10]. In order to explore antidiabetic chemical constituents of a daily vegetable for Bai nationality, O. acuminata var. acuminata was selected for this study. However, except for a preliminary analytical investigation by HPLC-ESI-MS, the phytochemical and antidiabetic ingredients of this vegetable have not been reported so far [11].
In this study, the first study of phytochemistry on O. acuminata var. acuminata was conducted and 41 compounds including six new diarylheptanoids (Otteacumienes A-F, 1-6) and 35 known ones possessing diverse diarylheptanoid, flavone, sesquiterpenoid, coumarin, lignan, and polyacetylene skeletons (7-41) were obtained ( Fig. 1). Structurally, the six new diarylheptanoids could be classified into three different types: diarylether, biaryl, and linear types of diaryheptanoids. Being distinct from diarylheptanoids reported from families Officinarum, Katsumadai, Blepharocalyx, Zingiberaceae, and Betulaceae [12], these structures in this study are characterized by low degree of oxidation. Bioactive study of compounds 3-8 showed substantial inhibitory effects on α-glucosidase but negligible effects on PTP1B, suggesting that they might be selective inhibitors of α-glucosidase. In addition, the molecular docking studies implied that different types diarylheptanoids are binding to the different sites of α-glucosidase, and the phenolic hydroxyl groups on diaryheptanoids might play a key role for their inhibitory activity.

Result and discussion
The DCM fraction of the EtOAc extract of O. acuminata var. acuminata was subjected to MCI-gel column, silica gel column chromatography, RP-C 18 column

Structural identification of compounds
Otteacumiene A (1) was isolated as a yellow crystal and its molecular formula was determined as C 19  Besides 16 carbon atoms of two benzene ring and two carbon-carbon double bonds, the remaining three carbon atoms were attributed to be two methylene (C-7 and C-13) and an oxygenated methine (C-8) according to the 13 C-NMR and DEPT spectra ( Table 2). The above characteristic signals implied that 1 could be a diaryheptanoid derivatives [14]. In the 2D NMR spectra, the key 1 H-1 H COSY correlations of H-7 (δ H 2.34, d, J = 4.5 Hz)/H-8/H-9/H-10/H-11/H-12/H-13 (δ H 3.51, m), together with the obvious HMBC correlations from H 2 -7 to C-3/C-5 and H 2 -13 to C-3′/C-5′/C-11 confirmed the presence of oxygenated unsaturated heptane chain and the connection of two benzene ring. The linkage of C-2 and C-1′ through an oxygen atom was elucidated by the downfield chemical shift of C-1 (δ C 145.0), C-1′ (δ C 157.6), and C-2 (δ C 152.3), the key HMBC correlations from 1-OH to C-1/C-2/C-6, together with the degrees of unsaturation. (Fig. 2). Finally, the absolute configuration of C-8 was undoubtedly determined to be 8S by X-ray diffraction analysis using Cu Kα radiation. [Flack parameter = 0.08(4)] (Fig. 3, CCDC 2156830).
Otteacumienes B (2) was isolated as yellow oil. The molecular formula was determined to be the same as 1 by HREIMS. Detailed analysis of the 1 H and 13 C NMR spectra indicated that the structure of 2 was similar with that of 1 (Tables 1, 2). For 2, the main difference from 1 was ascribed to the position of carbon-carbon bond (Δ 8,9 and Δ 10,11 ) and hydroxyl (at C-12) in the heptane chain as deduced by the 1 (Tables 1,  2). The most obvious differences of 3 compared to that of 1 lie in the appearance of an additional methoxy signal at δ H 3.20/δ C 57.4 as well as the absence of signals for a double bond in the heptane chain. These deductions were confirmed by downfield chemical shifts C-7 (δ C 87.5) and C-8 (δ C 75.0), the HMBC correlations from H-7 to C-3/C-4/C-5/C-9 and H-13 to C-3′/C-4′/C-5′/C-11, and COSY correlations of H-7/H-8/H-9/H-10/H-11/H-12/H-13 ( Fig. 2). In addition, the crystals for single-crystal X-ray diffraction (Fig. 3, CCDC 2155136) were obtained, which clarified the relative configuration and the racemic nature of 3 with the crystal space group P2/n. After attempts with various chiral columns and conditions of mobile phase, the chiral separation of 3 was achieved on a chiral-phase HPLC apparatus using a DAICEL CORPO-RATION semi-preparative column (Fig. S2). To further determine the absolute configurations of enantiomers, quantum-chemical calculation method was used, which eventually assigned the absolute configurations of (+)-3 and (−)-3 to be 7S, 8S and 7R, 8R, respectively (Fig. 4).   Table 1). The 13 C NMR and DEPT spectra displayed 19 carbon signals attributing to seven quaternary carbons (one carbonyl), eight methine, and four methylene. The evidences mentioned above, conjugated with a pair of unusual high field aromatic quaternary carbon signals at δ C 128.3/ 126.6 (C-2/C-2′) suggest that 4 could be a biaryl type cyclic diaryheptanoid derivative (Tables 1, 2 8 Hz) and HMBC correlations from H 2 -7 to C-3/C-5/C-9, from H-10 to C-8/C-12, and from H 2 -13 to C-3′/C-4′/C-5′ established its biaryl type cyclic diaryheptanoid architecture. Then, the connection between C-2 and C-2′ was confirmed by the degrees of unsaturation together with the HMBC correlations of H-3/C-2′ and H-3′/C-2 (Fig. 2). Therefore, the structure of 4 was elucidated.
Compound 6 was also isolated as green oil, showed a molecular ion at m/z 293.1547 [M -H]in the HRESIMS (calculated 293.1542), which correlates to the molecular formula C 20 H 22 O 2 . The 1 H and 13 C NMR spectra were similar with those of 5 (Tables 1, 2) except for an extra methoxy signal (δ H 3.75, δ C 55.3) in 6. And this methoxyl was deduced to be located at C-1′ by its HMBC correlation with C-1′ (δ C 158.9). Therefore, its structure was established as the 1'-O-methylated 5 and named otteacumiene F.

α-Glucosidase and PTB1B inhibitory activity
To explore the antidiabetic chemical constituents from O. acuminata var. acuminata, the α-glucosidase and PTP1B of inhibitory activities of diaryheptanoids 1-8 were evaluated. The results revealed that these compounds exhibited different levels of inhibitory activity ranging from 38.29% to 103.55% at 50 μM (Table 3), in which 3-8 with inhibition rates more than 50.0% were screened for their IC 50 values (acarbose as a positive control). Interestingly, all these compounds exhibited more potential inhibitory activity with IC 50 values of 3.81-26.44 μM (Table 3). Especially, 5 represented most effective inhibitor with 60 times more potent than that of acarbose (228.95 μM), the first-line drug for diabetes treatment. It's notable that these diaryheptanoids exhibited negligible effects on PTP1B (Table S11), suggesting that these active ingredients may serve as selective inhibitors on α-glucosidase.

Molecular docking studies
To further explore the potential antidiabetic mechanism of the diarylheptanoids, the molecular docking studies were performed by using PyMol program. Compounds 4, 5, and 8 were selected as representative structures of diarylether, linear, and biaryl types of diaryheptanoids for molecular docking against α-glucosidase. The results showed that all the three types of diaryheptanoids act with different cavities mode with that of acarbose [48] which may have contributed to their substantial α-glucosidase inhibitory activities. The molecular docking study for acarbose against α-glucosidase indicated that this first-line medicine formed six hydrogen bonds with ASP-242 (2.9 Å), SER-242 (3.5 Å), GLN-279 (2.8 Å), ARG-422 (2.8 Å), GLU-411 (2.8 Å), and AGR-315 (2.9 Å), respectively. Notably, the 1′-OH of 5 formed three hydrogen bonds with SEP-241 (2.9 Å, 3.2 Å) and ARG-422 (3.0 Å), which might be the reason for its superior activity in contrast to 4 and 8. Correspondingly, 1-OH and 1′-OH of 4 formed two hydrogen bonds with TRY-158 (2.7 Å, 2.8 Å) and 1-OH of 8 formed a hydrogen bond with MET-70 (3.3 Å). Although 4, 5, and 8 in the docking process tended to combine with the same cavity, the difference in the ability to form hydrogen bonds with amino acid residues might be the reason for their different activities (Fig. 5).

Conclusion
To the best of our knowledge, this is the first study on the phytochemistry of O. acuminata var. acuminata and their antidiabetic activity. Totally, 41 metabolites ingcluding eight diarylheptanoids (six new ones), eight phenylpropanoids, four lignans, eight coumarins, seven flavonoids, two polyacetylenes, a sesquiterpenoid, two alkaloids, and a vanillin were characterized in this study. Among them, 3 was obtained as a pair of enantiomers whose absolute configurations were determined by calculated ECD method after chiral separation. The biological activity studies displayed that 3-8 exhibited substantial inhibitory activity on α-glucosidase as well as negligible effects on PTP1B, which indicated that these diarylheptanoids might be selective inhibitors of α-glucosidase. Notably, compound 5 was 60-fold

General experimental procedures
IR spectra were measured on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. Optical rotations were recorded on a JASCO P-1020 polarimeter. UV spectra were obtained with a Shimadzu UV-2401PC spectrometer. 1D and 2D NMR spectra were performed on a Bruker DRX-600 spectrometer using TMS as an internal standard. The chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HREIMS and HRESIMS analysis were obtained from Waters Xevo TQS and Agilent G6230 TOF mass spectrometers, respectively. Single-crystal X-ray diffraction data were exhibited on a Bruker D8 QUEST diffractometer. Semi-preparative HPLC was performed on a Waters 1525 HPLC with a ZORBAX SB-C 18

Extraction and isolation
The dried samples of O. acuminata var. acuminata (20.0 kg) were crushed and extracted four times with methanol for two days each time, and the solvent was recovered under reduced pressure to obtain a crude extract (2.5 kg). The crude extract was eluted with 90% methanol through macroporous resin to obtain 200 g of eluted fractions. The 90% methanol eluent was applied to silica gel column chromatography eluted with dichloromethane (DCM), to afford fraction O-DCM (30.5 g).
Fraction O-DMC (30.5 g) was separated over an MCIgel column (MeOH-H 2 O from 6:4 to 10:0, v/v) to obtain six fractions from small to large polarities Fr. A-F, which were successively on purified by macroporous resin, silica gel, MCI-gel, RP-C 18 , and preparative or semi-preparative HPLC chromatographic metheds to give the 41 isolates (detailed process, Fig. S1).

α-Glucosidase and PTB1B inhibitory activities assay
The α-glucosidase and PTB1B inhibitory activity were conducted according to the previous reports with slight modifications [49,50]. Briefly, in the α-glucosidase inhibitory assay, after 50 min incubation at 37 °C, the absorbance value at 405 nm was detected and acarbose was used as positive control. While in the PTB1B inhibitory assay, after addition of phosphate-based detection reagent then incubation at 30 °C for 20 min, and absorbance was measured at 620 nm and suramin was used as positive control. The inhibition percentage was calculated as follows: inhibition rate (%) = (E -S)/E × 100% (E is the OD of the control and S is the OD of the sample) and IC 50 (50% concentration of inhibition) was calculated by Reed and Muench method.

Molecular docking studies
To explore the structure-activity relationship, the molecular docking studies was conducted according to the previous reports with slight modifications [51]. In brief, the AutoDock and PyMol software was used to blind docking between 3D structure of α-glucosidase which is downloaded from RCSB PDB website (PDB ID: 3A4A) and compound ligands.